The long-term goal of proposed research is to understand the biochemistry and cell biology of protein folding in eukaryotic cells. The proposed research will focus on folding events as they occur at the ribosome during synthesis of a polypeptide and will examine the role of molecular chaperones in the folding process. The conceptual framework required to understand the folding of proteins as they emerge from the ribosome originates from the principal investigator's previous work, which indicates that folding in the eukaryotic cytosol is mediated by a highly organized chaperone machinery that is coupled to translation. Dr. Frydman's working hypothesis is that the newly translated polypeptides are guided to their final conformation through a sequential and highly coupled chaperone pathway. Results from Dr. Frydman's laboratory also indicate that different subsets of cellular proteins exhibit different chaperone requirements. For the model proteins that are the focus of the proposed research, the folding pathway appears to involve two classes of chaperones, namely small chaperones, such as the Hsc70 proteins and the GIM/prefolclin complex, which act by stabilizing extended polypeptides, and the chaperonin TRiC, which by virtue of its ring-like structure creates an environment that is favorable for polypeptide folding. The objective of this proposal is to elucidate the mechanism by which chaperones mediate the folding of newly synthesized proteins in eukaryotic cells. The general strategy is to combine in vitro and in vivo approaches to obtain mechanistic and functional insights into the role of chaperones in cellular folding. Specifically the principal investigator will: 1) Define the chain-length dependence of the interactions of nascent polypeptides with chaperone proteins; 2) Assess the requirement of molecular chaperones in the folding of newly synthesized polypeptides; 3) Determine the mechanisms that mediate the recruitment of chaperone components to the ribosome-bound nascent chain; 4) Define the substrate spectrum of the cytosolic chaperones in intact cells.